N-phosphonomethylglycine, commonly referred to as glyphosate, is an important agronomic chemical. Glyphosate inhibits the enzyme that converts phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid. Inhibition of this enzyme (5-enolpyruvylshikimate-3-phosphate synthase; referred to herein as “EPSP synthase”) kills plant cells by shutting down the shikimate pathway, thereby inhibiting aromatic acid biosynthesis.
Since glyphosate-class herbicides inhibit aromatic amino acid biosynthesis, they not only kill plant cells, but are also toxic to bacterial cells. Glyphosate inhibits many bacterial EPSP synthases, and thus is toxic to these bacteria. However, certain bacterial EPSP synthases may have a high tolerance to glyphosate.
Plant cells resistant to glyphosate toxicity can be produced by transforming plant cells to express glyphosate-resistant EPSP synthases. A mutated EPSP synthase from Salmonella typhimurium strain CT7 confers glyphosate resistance in bacterial cells, and confers glyphosate resistance on plant cells (U.S. Pat. Nos. 4,535,060, 4,769,061, and 5,094,945). Thus, there is a precedent for the use of glyphosate-resistant bacterial EPSP synthases to confer glyphosate resistance upon plant cells.
An alternative method to generate target genes resistant to a toxin (such as an herbicide) is to identify and develop enzymes that result in detoxification of the toxin to an inactive or less active form. This can be accomplished by identifying enzymes that encode resistance to the toxin in a toxin-sensitive test organism, such as a bacterium.
Castle et al. (WO 02/36782 A2) describe proteins (glyphosate N-acetyltransferases) that are described as modifying glyphosate by acetylation of a secondary amine to yield N-acetylglyphosate.
Barry et al. (U.S. Pat. No. 5,463,175) describes genes encoding an oxidoreductase (GOX), and states that GOX proteins degrade glyphosate by removing the phosphonate residue to yield amino methyl phosphonic acid (AMPA). This suggests that glyphosate resistance can also be conferred, at least partially, by removal of the phosphonate group from glyphosate. However, the resulting compound (AMPA) appears to provide reduced but measurable toxicity upon plant cells. Barry describes the effect of AMPA accumulation on plant cells as resulting in effects including chlorosis of leaves, infertility, stunted growth, and death. Barry (U.S. Pat. No. 6,448,476) describes plant cells expressing an AMPA-N-acetyltransferase (phnO) to detoxify AMPA.
Phosphonates, such as glyphosate, can also be degraded by cleavage of C—P bond by a C—P lyase. Wacket et al. (1987) J. Bacteriol. 169:710-717) described strains that utilize glyphosate as a sole phosphate source. Kishore et al. (1987) J. Biol. Chem. 262:12164-12168 and Shinabarger et al. (1986) J. Bacteriol. 168:702-707 describe degradation of glyphosate by C—P Lyase to yield glycine and inorganic phosphate.
While several strategies are available for detoxification of toxins, such as the herbicide glyphosate, as described above, new activities capable of degrading glyphosate are useful. Novel genes and genes conferring glyphosate resistance by novel mechanisms of action would be of additional usefulness. Single genes conferring glyphosate resistance by formation of non-toxic products would be especially useful.
Further, genes conferring resistance to other herbicides, such as the sulfonylureas or imidazolinones, are useful. The sulfonylurea and imidazolinine herbicides are widely used in agriculture because of their efficacy at low use rates against a broad spectrum of weeds, lack of toxicity to mammals, and favorable environmental profile (Saari et al. (1994) p. 83-139 in: Herbicide Resistance in Plants: Biology and Biochemistry. S. Powles and J. Holtum eds. Lewis Publishers, Inc., Boca Raton, Fla.). These herbicides act by inhibiting acetohydroxyacid synthase (AHAS, also known as acetolactate synthase) and thereby preventing the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine.
Current methods of herbicide tolerance confer upon a plant tolerance to herbicides with a particular target or mode of action. However, repeated and extensive use of herbicides with a single mode of action can result in the selection of tolerant weed species (Saari et al., supra). Crop plants which are resistant to more than one class of herbicides (with different modes of action) provide growers with flexibility in weed control options and are useful in preventing/managing the emergence of resistant weed populations. Plants containing a single trait that conferred tolerance to more than one class of herbicide would be particularly desirable. Thus, genes encoding resistance to more than one class of herbicide are useful.
Thus, methods that result in degradation of herbicides to non-toxic forms are desired. Further, methods that achieve sufficient degradation to allow cells to grow in otherwise toxic concentrations of herbicide (“herbicide resistance”) are desired. Methods that confer “herbicide resistance” through the expression of a single protein would be preferred, since expression of a single protein in a cell such as a plant cell is technically less complex than the expression of multiple proteins. Further, in some instances, methods for conferring herbicide resistance that are compatible with, and/or improve the efficacy of other methods of conferring herbicide resistance, are desirable.